A. Transgenic Strains
Transgene
syIs187
mhIs9
wyEx806
huIs7
muIs49
sEx10067
otIs198
nsIs230
deIs12
muIs53
nsEx4592
nsEx4584-5
nsEx4589-90
nsEx4591
nsEx4586-8
nsSi2-3
nsIs346
nsIs495
Constructs
POPTOP::HIS-24-mCherry
lin-17::GFP
lin-44::GFP + odr-1p::GFP
hsp-16.2p::ΔN-BAR-1, dpy-20(+), mec-7::GFP
egl-20::GFP, unc-22(+)
hsp-1p::GFP
hsp-16.2p::NLS-mCherry
hsp-16.41p::mCherry
10.8kb-bar-1p::GFP
hsp-16.2p::egl-20 + unc-22(as)
pGO80[2.5kb wrm-1p::GFP], a gift of G. Oikonomou, 25 ng/L + unc-119(+), 75
ng/L
pMJK61[mig-24::mig-5B] (10 ng/L) + pEB30[lag-2p::mCherry] (25 ng/L) +
odr-1p::RFP (25 ng/L) + L4440, 40 ng/L
pMJK62[mig-24::mig-1B] (50 ng/L) + pEB30[lag-2p::mCherry] (25 ng/L) +
odr-1p::RFP (25 ng/L)
pMJK62[mig-24::mig-1B] (25 ng/L) + pEB30[lag-2p::mCherry] (25 ng/L) +
odr-1p::RFP (25 ng/L)
pMJK60[mig-24::HSF-1] (1 ng/L) + pEB30[lag-2p::mCherry] (25 ng/L) + odr1p::RFP (25 ng/L) + pBluescript, 49 ng/L
MosSCI integrants of pOG124[hsf-1p::HSF-1(R145A)-GFP]
UV/TMP integration of mhEx127[5kb mig-5p::mig-5A-GFP + unc-119], a gift of
M. Herman.
lin-48p::mCherry (25 ng/L) + pBluescript (75 ng/L), a gift of L. Kutscher.
nsEx3669-72
pJAZ042[mig-24p::let-70 C. briggsae cDNA silently point mutated to avoid RNAi]
+ unc-119(+)
nsEx3081
mig-24p::rde-1 cDNA::SL2::mCherry + lag-2p::mCherry
nsEx2866,
pJAZ002 (let-70(1.5kb)p::GFP) + unc-119(+)
nsEx2864-5
pJAZ001 (let-70(1.5kb)p::let-70::GFP + unc-119(+)
nsEx3683-4
pJAZ038[mig-24p::let-70(C35S) C. briggsae cDNA] + unc-119(+)
nsEx4197,
pJAZ117[rbx-1p::GFP] + unc-119(+)
nsEx3950,
52-53, 94
pJAZ068[ubq-1p::ubq-1 first monomer::GFP] + unc-119(+)
sEx10785
cul-3p::GFP + pCeh361 (dpy-5 (+))
sEx10148
rpn-3p::GFP + pCeh361 (dpy-5 (+))
nsEx5608
let-70-gfp fosmid (WRM0627aA07)
nsIs524-5
pJAZ002-delHSE (let-70(HSE)p::GFP) + unc-119(+)
nsEx5153
mig-24p::btbd-2(1ng/L) + lag-2p::mCherry(30ng/L) + pBluescript(70ng/L)
nsEx5154
mig-24p::btbd-2 (1ng/L) + lag-2p::mCherry (30ng/L) + unc-119(+) (70ng/L)
B. Plasmids and plasmid construction
Construct
pMJK60
Description
mig-24p::hsf-1
pMJK61
mig-24p::mig-5B
pMJK62
mig-24p::mig-1B
pJAZ001
let-70p::GFP in
pPD95_75
Notes
A ~1.2kb mig-24 promoter, a gift of E. Blum,
was amplified as a PstI/XmaI fragment using
oligos 5’ACACTGCAGTTTATCAGTTATCAGCAA
GCAGAGAAATG -3’ and 5’ACACCCGGGCGCCATTTTAATAAAATT
GTGTAAGATG -3’ and subcloned into
pTB4[vap-1::hsf-1::unc-54 3’UTR]
mig-5B cDNA, a gift of H. Korswagen, was
amplified as an XmaI/SalI fragment using
oligos 5’AGTCCCGGGATGGAGCCGCCATGCAC3’ and 5’ATCGTCGACCTACTGTTCTCCCCGTCGA
AATC-3’ and subcloned into pMJK60.
mig-24 promoter was amplified as an
FseI/AscI fragment using oligos 5’ACTGGGCCGGCCTTTATCAGTTATCAGC
AAG-3’ and 5’CCATGGCGCGCCTCATTTTAATAAAATT
GTGTAAG-3’ and subcloned into pSM-SL2mCherry, a gift of C. Bargmann. mig-1B
cDNA, on pJRK135, also a gift from C.
Bargmann, was amplified as an AscI/EcoRI
fragment using oligos 5’TGAGGCGCGCCATGGGACCATTTCGTG
GTTAC-3’ and 5’ACTGAATTCTCAAATCATATTATTAGTT
CGAAACGTC-3’ and subcloned into this
plasmid.
N2 genomic sequence was amplified using
primers: 5’-
pJAZ002
let-70p::GFP in
pPD95_75
pJAZ005
siah-1 RNAi in L4440
pJAZ006
let-70 RNAi in L4440
pJAZ008
let-70 3’UTR RNAi in
L4440 (RNAi C)
pJAZ009
nhr-67/let-70 RNAi in
L4440
pJAZ012
set-16/let-70 RNAi in
L4440
pJAZ013
nhr-67 RNAi in L4440
GATTGGGCATGCTTTCCATATCTCTTG
and 5’GTTAGGGTACCCACATAGCGTACTTTTG
CGTCC, and ligated into pPD95.75 (Andrew
Fire) as an SphI/KpnI fragment.
N2 genomic sequence was amplified using
primers: 5’GATTGGGCATGCTTTCCATATCTCTTG
and 5’CTTTTGGTACCCATGTTGGTGTTCTGAT
TTGCTG, and ligated into pPD95.75 (Andrew
Fire) as an SphI/KpnI fragment.
cDNA library was amplified using primers: 5’GCGCGGCCGCAAACGATGGCCAGAGTT
CAG and 5’GCGCGTCGACTCCAAGGTTTCCGTTTTC
TG. This XbaI/XmaI fragment was ligated into
L4440 (Julie Ahringer).
The entire let-70 cDNA sequence was
amplified from a cDNA library using 5’GCGCTCTAGATCCATTGGCAAGCTACG
ATT and 5’GCGCGGTACCCCATTCTCTCGCCAATTG
AT. This XbaI/KpnI fragment was ligated into
L4440 (Julie Ahringer).
N2 genomic sequence was amplified using 5’GGCGGCCGCGAGGCTAACACCATTCAT
ACAAGA and 5’GGGTCGACATGCAGTGGGCTCTTCAGT
AATTTATTCAGGATTCTCAGACC. This
NotI/SalI fragment was ligated into L4440.
N2 genomic sequence was amplified using 5’GGCCTCTAGAGGATCCTCTCCTAATTCG
CC and 5’GGCCCCCGGGTGTTGTGGTGGCTTGAA
CAT. This XbaI/XmaI fragment was ligated
into an L4440 already containing let-70 RNAi
between sites HindIII/XhoI.
N2 genomic sequence was amplified using 5’GCGCTCTAGATGCAATCATAAAGCAGC
GTCand 5’GCGCCCCGGGGCGTCACTTGATCTGAG.
This XbaI/XmaI fragment was ligated into an
L4440 already containing let-70 RNAi
between HindIII and XhoI (pJAZ021).
N2 genomic sequence was amplified using 5’-
pJAZ015
set-16 RNAi in L4440
pJAZ018
tir-1 RNAi in L4440
pJAZ019
tir-1/let-70 RNAi in
L4440
pJAZ021
let-70 RNAi in L4440
pJAZ024
rbx-1/let-70 RNAi in
L4440
pJAZ026
swd-2.2/let-70 RNAi in
L4440
pJAZ027
mCherry/let-70 RNAi in
L4440
GGCCTCTAGAGGATCCTCTCCTAATTCG
CC and 5’GGCCCCCGGGTGTTGTGGTGGCTTGAA
CAT. This XbaI/XmaI fragment was ligated
into L4440.
N2 genomic sequence was amplified using 5’GCGCTCTAGATGCAATCATAAAGCAGC
GTCand 5’GCGCCCCGGGGCGTCACTTGATCTGAG.
This XbaI/XmaI fragment was ligated into
L4440.
N2 genomic sequence was amplified using 5’GCGCTCTAGAATCGGATACGATGAAGC
TGG and 5’GCGCCCCGGGCCGATGAGTAATCAGCA
GCA. This XbaI/XmaI fragment was ligated
into L4440.
N2 genomic sequence was amplified using 5’GCGCTCTAGAATCGGATACGATGAAGC
TGG and 5’GCGCCCCGGGCCGATGAGTAATCAGCA
GCA. This XbaI/XmaI fragment was ligated
into an L4440 already containing let-70 RNAi
between HindIII and XhoI (pJAZ021).
cDNA library was amplified using 5’GCGCAAGCTTTCCATTGGCAAGCTACG
ATT and 5’GCGCCTCGAGCCATTCTCTCGCCAATTG
AT, and inserted into L4440 using
HindIII/XhoI.
N2 genomic sequence was amplified using 5’GCGCTCTAGAATGGCCCAAGCAAGCGA
CAG and 5’GCGCCCCGGGTTAGTGTCCGTACTTTTG
GA. This XbaI/XmaI fragment was ligated into
an L4440 already containing let-70 RNAi
between HindIII and XhoI (pJAZ021).
N2 genomic sequence was amplified using 5’GCGCTCTAGATTTGTTAATTCCGGGTTT
GC and 5’GCGCCCCGGGCGGAGCTTCCCACCATA
ATA. This XbaI/XmaI fragment was ligated
into an L4440 already containing let-70 RNAi
between HindIII and XhoI (pJAZ021).
N2 genomic sequence was amplified using 5’GCGCTCTAGAAAGGGCGAGGAGGATAA
pJAZ030
rpn-3 RNAi in L4440
pJAZ031
rpn-3/let-70 RNAi in
L4440
pJAZ033
rbx-1 RNAi in L4440
pJAZ035
lin-29 RNAi in L4440
pJAZ036
lin-29/let-70 RNAi in
L4440
pJAZ038
mig-24p::let-70 briggsae
C85S
pJAZ040
mig-24p::let-70 elegans
cDNA
CAT and 5’GCGCCCCGGGTTGACCTCAGCGTCGTA
GTG. This XbaI/XmaI fragment was ligated
into an L4440 already containing let-70 RNAi
between HindIII and XhoI (pJAZ021).
N2 genomic sequence was amplified using 5’GCGCTCTAGAACAATGATCTTGCCCGCT
AC and 5’GCGCCCCGGGGGACAGGGATCTCGATT
TGA. This XbaI/XmaI fragment was ligated
into an L4440 vector.
N2 genomic sequence was amplified using 5’GCGCTCTAGAACAATGATCTTGCCCGCT
AC and 5’GCGCCCCGGGGGACAGGGATCTCGATT
TGA. This XbaI/XmaI fragment was ligated
into an L4440 already containing let-70 RNAi
between HindIII and XhoI (pJAZ021).
N2 genomic sequence was amplified using 5’GCGCTCTAGAATGGCCCAAGCAAGCGA
CAG and 5’GCGCCCCGGGTTAGTGTCCGTACTTTTG
GA. This XbaI/XmaI fragment was ligated into
an L4440 vector.
N2 genomic sequence was amplified using 5’GCGCTCTAGAAGAGCACAGATTTTTGA
ACGAAG and 5’GCGCCCCGGGATTTAATTTTACAGCCG
ATGGGT. This XbaI/XmaI fragment was
ligated into an L4440 vector.
N2 genomic sequence was amplified using 5’GCGCTCTAGAAGAGCACAGATTTTTGA
ACGAAG and 5’GCGCCCCGGGATTTAATTTTACAGCCG
ATGGGT. This XbaI/XmaI fragment was
ligated into an L4440 already containing let-70
RNAi between HindIII and XhoI (pJAZ021).
pJAZ042 was mutagenized using Quikchange
primers: 5’ATTAACTCGAACGGATCGATTTCCCTCG
ATATTCTCCGATCACA and 5’TGTGATCGGAGAATATCGAGGGAAATC
GATCCGTTCGAGTTAAT using sitedirected mutagenesis kit (Stratagene)
cDNA library was amplified using 5’GCGCGGTACCGGTAGAAAAAATGGCTC
pJAZ042
mig-24p::let-70 briggsae
point mutated rescue
pJAZ060
6xHis::let-70 in pET28b(+)
pJAZ068
ubq-1p::ubq-1::GFP
pJAZ117
rbx-1p::GFP
pJAZ151
cul-1/let-70 RNAi
pJAZ119
let-70(HSE)::GFP
pJAZ200
mig-24::btbd-2 cDNA
TCAAAAGAATCCA and 5’GCGCGAATTCCAGACGCTCTTCAGTAA
TTT. The KpnI/EcoRI fragment was ligated
into a mig-24p::mCherry vector.
let-70 cDNA carrying point mutations and
flanked by KpnI/EcoRI was engineered via
GeneArt Gene Synthesis (Life Technologies).
The fragment was digested and inserted into a
mig-24p::mCherry vector using KpnI/EcoRI.
pJAZ040 was used as a template for
amplification using the primers 5’GCGGGATCCATGGCTCTCAAAAGAATC
CAG and 5’GCGGCCGCTCACATAGCGTACTTTTGCG
The fragment was ligated into pET28b(+)
(Novagen) using BamHI and NotI.
N2 genomic sequence was amplified using
primers 5’GCGCGCATGCCAGTTGCATGACGAGAA
AAAAGA and 5’GGTACCCCAATGTCTCCTCCGCGAAGA
CGAAGAACG. The SphI/KpnI fragment was
cloned into pPD95.75.
N2 DNA was amplified using primers 5’CGCGGCATGCATGCTTATCATGCCTTTC
TAGGTAAA and 5’CGCGGGTACCGGTTTCTGGAAAAAATA
CAAATGAGAATAA. The SphI/KpnI
fragment was inserted into pPD95.75 (Andrew
Fire).
C. elegans cDNA was amplified using primers
5’CGCGTCTAGAGAAGACGGTCGAGCAGA
ATC and 5’CGCGCCCGGGGCATGAATCCTGGCTCA
TTT. The XbaI/XmaI fragment was inserted
into pJAZ021.
pJAZ002 was amplified using primers 5’ATTTTATTAGTGTGACGTAACAGTTCAC
CACGAAGG and 5’TTACGTCACACTAATAAAATAACTAAC
GATGAGAAATGATG. The fragment was
DpnI digested and transformed.
C. elegans cDNA was amplified using primers
5’TAAAATGGCGGTACCATGAGCTCGGAA
AATGGAG and 5’CGCTCAGTTGGAATTCTCACTCGGCTGC
TGTATAG. The fragment was Gibson cloned
into mig-24p::mCherry digested with
SacII/XmaI.